Of the various reagents used to transfect cells with bioactive agents such as nucleic acids, those based on liposome mediated delivery are widely acknowledged to be the most effective. This is due mostly to their efficiency and ease of use. Liposomes are artificially prepared spherical vesicles made of lipid bilayer. To deliver the molecules to sites of action, the lipid bilayer can fuse with other bilayers such as a cell membrane, thus delivering the liposome contents inside a cell.
Liposomes are used for drug delivery due to their unique properties. A liposome encapsulates a region of aqueous solution inside a hydrophobic membrane; dissolved hydrophilic solutes cannot readily pass through the lipids. Hydrophobic chemicals can be dissolved into the membrane, and in this way liposome can carry both hydrophobic molecules and hydrophilic molecules. Liposomes can be combined with bioactive agents such as drugs, nucleic acids, peptides etc., and used to deliver these agents for the regulation of a cells biochemical pathway. This opens possibilities for new treatments of diseases.
Gershon et al. (Gershon H, et al. Mode of formation and structural features of DNA-cationic liposome complexes used for transfection. Biochemistry. 1993, 32:7143-7151) state that nanoparticles which are formed between cationic liposomes and nucleic acids represent an efficient vehicle for delivery of DNA and RNA into cell. Cationic liposomes bind initially to DNA molecules to form clusters of aggregated vesicles along the nucleic acids. At a critical liposome density, two processes occur, DNA-induced membrane fusion and liposome-induced DNA condensation. The DNA condensation leads to the formation of condensed structures which can be completely encapsulated with the fused lipid bilayers in a fast, highly cooperative process.
For delivery of negatively charged nucleic acid, cationic lipids are the most effective transfection agents. Cationic lipids represent a promising class of synthetic materials for DNA delivery. To date, there are several commercialized cationic lipids but the number of cationic lipids for safe and effective delivery of genes is still limited.
In “Cationic liposomes for gene therapy” (Miller A D, Angew Chem Int Ed. 1998, 37:1768-1785) most of the commonly used and commercially available transfection agents are described. However, conventional lipid synthesis typically requires individually optimized, multiple-step synthesis, including time-intensive procedures such as chemical protection and deprotection, use and removal of catalysts, solvent exchanges and purification.
WO 01/42200 describes examples of cationic amphiphilic compounds and their use in pharmaceutical compositions as transfection agents. The compounds disclosed in this document are made in a time consuming multiple-step synthesis.
Cationic lipids need to be combined with natural phospholipids (referred to as helper lipids) to form liposomes that can be more efficiently incorporated into cell membranes. By combining liposomes with DNA or drugs, which alone cannot diffuse through the membrane of the target cell, they can be (indiscriminately) delivered past the lipid bilayer. The use of liposomes for transformation or transfection of DNA into a host cell is known as lipofection.
Although liposomal reagents represent the state of the art with respect to cell transfection agents, they have the following drawbacks:
1. Many cell lines (such as primary cells) cannot be effectively transfected at the moment, even with liposomal reagents.
2. They are relatively difficult and expensive to synthesise, often resulting in high price for end-users.
As a consequence of the second point, many laboratories use less efficient, cheaper alternatives for transfection (e.g. calcium phosphate). There is a concrete requirement for new transfection agents that are easy to synthesize and which have good transfection yields for a wide variety of cell types. As an alternative, it would be helpful to dispose an easy combinatorial synthesis of transfection agents allowing the production of a variety of different compounds.